The proposed research is a biochemical and genetic study of mitochondrial biogenesis in Neruosporo. The long term goals of this study are to define the roles of the nuclear and mitochondrial genetic systems and to understand how these roles are coordinated and modulated. Specific aims fall in two areas (a) mitochondrial iibosome assembly, a process that requires interaction between the nuclear and mitochondrial genetic systems, and (b) mitochondrial plasmids, candidate mobile elements that are found in certain Neurospora strains. In previous work. we identified and characterized nuclear and extranuclear Neurosporo mutants that affect mitochondrial ribosome assembly. In the proposed research, we would use the nuclear mutants to isolate specific nuclear genes that function in mitochondrial ribosome assembly. Initial objectives are to isolate nuclear genes that encode mitochondrial ribosomal proteins, to characterize these genes, and to study their regultion. An ancillary goal in this part of the research is to develop further the technology for the isolation and analysis of Neurospora nuclear genes. In previous work, we showed that one mitochondrial ribosomal protein, S-5 (Mr = 52,000), is synthesized in the mitochondria and obtained evidence that this protein is required for the assembly of mitochondrial small ribosomal subunits. Recent DNA sequence analysis suggests that S-5 is encoded within the mitochondrial large rRNA intron. As the only mitochondrially-synthesized mitochondrial ribosomal protein, S-5 is in a position to play a key role in regulation. We propose to continue to study the role of S-5 in mitochondrial ribosome assembly. Finally, we have identified and characterized Neurospora mitochondrial plasmids that are not homologous to the standard mtDNA. The DNA sequence of the 3.6 kb plasmid present in the Mauriceville-lc strain of Neurospora crassa shows that it contains an open reading frame that could encode a protein as long as 710 amino acids. In addition, several characteristics of the plasmid suggest that it may belong to a class of mobile genetic elelments of the type that were or are the progenitors of the predominant group of mtDNA introns (Group I). We propose to continue to study Neurospora mitochondrial plasmids, to identify and charactrize the plasmid-encoded protein(s), and to test directly the ability of the plasmids to function as mobile elements.